A new simple, rapid and selective high performance thin layer chromatography (HPTLC) method is developed for the quantitation of acyclovir during in vitro skin permeation studies. Separation of guinea pig skin proteins and acyclovir was achieved by employing a mobile phase consisting of chloroform-methanol-ammonia (15:9:4, v/v/v) on precoated silica gel 60F254 aluminum plates. Densitometnic analysis was carried out at 255 nm. The limit of detection and quantification were 30 and 50 ng, respectively. The calibration curve was linear in the range of 10-20 μg/ml (r=0.9965). The relative standard deviation for a sample of concentration 100 μg/ml were 1.15 and 2.85 for system and method precision, respectively. Intraday and interday variation studies gave an average 0.763 and 0.463% relative standard deviation for the three levels tested. Average recoveries of 101.8 and 100.1% were recorded for two marketed preparations studied. The method was employed to optirmize topical liposomal gel formulation of acyclovir on basis of maximum skin permeation. Copyright (C) 2000 Elsevier Science B.V.